A fusion protein targeting stress granules for degradation and applications thereof

Abstract

The present application relates to a kind of fusion protein targeted to degrade stress granule and its application, belong to the field of biological medicine technology.The present application first provides a kind of fusion protein targeted to degrade stress granule, the fusion protein with G3BP1 protein as recognition module, with TRIM21 as degradation induction module, can realize the specific degradation of stress granule aggregation state.Further, the present application finds that when the NTF2L domain of G3BP1 protein is used as the recognition module, and the RING domain of TRIM21 is used as the degradation induction module, the degradation effect on the stress granule in aggregation state is the best, and has wide application prospect in the treatment of stress granule aggregation-mediated neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a fusion protein that targets and degrades stress particles and its applications. Background Technology

[0002] Stress granules (SGs) are a class of membraneless organelles that rapidly assemble in response to stresses such as oxidative stress, heat stress, or viral infection. They are primarily composed of mRNA and RNA-binding proteins (RBPs). The main functions of stress granules are to inhibit the initiation of mRNA translation, maintain mRNA stability, and regulate cell survival-related signaling pathways. Under normal physiological conditions, stress granules are usually rapidly deassembled after stress is relieved, allowing the cell's translation process to return to normal.

[0003] However, recent studies have shown that under pathological conditions, especially in neurodegenerative diseases, the dynamic assembly and disassembly of stress granules become unbalanced. Stress granules can form non-degradable pathological protein aggregates, which co-aggregate with known pathogenic proteins such as TAR DNA-binding protein-43 (TDP-43), FUS protein, and Tau protein (microtubule-associated protein), forming irreversible toxic protein aggregates. These aggregates interfere with normal cellular physiological functions and participate in the occurrence and development of diseases. Therefore, targeted regulation or degradation of pathological protein aggregates is a potential intervention strategy for neurodegenerative diseases.

[0004] Currently, there are significant deficiencies in the regulation techniques for stress particles, making it difficult to meet the needs of precise regulation. Specifically: (1) Gene silencing: such as RNA interference (RNAi) or the CRISPR-Cas9 system to knock down or remove key components of stress particles, such as GTPase activator protein SH3 domain-binding protein 1 (G3BP1) and cytotoxic particle-associated RNA-binding protein (TIA1). These methods often result in complete protein loss, which may disrupt normal mRNA homeostasis and translation regulation, and lack precision and reversibility. (2) Activation of degradation pathways: small molecule drugs can improve the efficiency of aggregate clearance, but they cannot achieve specific recognition of stress particles. They may be accompanied by the widespread degradation of non-target proteins, and excessive activation of the autophagy or proteasome system may also interfere with normal cellular metabolism. (3) Novel protein-targeted degradation tools: Proteolytic targeted chimeras (PROTACs) recruit target proteins to E3 ubiquitin ligases through bifunctional molecules, inducing their ubiquitination and degradation. This technology is effective in kinases and receptor proteins, but it depends on the target protein having natural small molecule ligands or ubiquitination-inducible domains. However, the core components of SG such as G3BP1 and TIA1 are non-enzymatic proteins with compact structures and lacking clear small molecule binding sites, making them difficult to adapt to this technology. (4) Regulatory studies based on ubiquitination mechanisms: Ubiquitin modification is involved in the formation and depolymerization of stress particles. For example, G3BP1 can be ubiquitinated by the 63rd lysine (K63) ubiquitin chain and recognized by valine-containing protein (VCP) to pull out the core structure of SG, triggering depolymerization. Studies have also shown that ubiquitin modification guides SG into the autophagy pathway through receptors such as scaffold protein p62, calcium-binding and coiled-coil domain (CALCOCO2).

[0005] The aforementioned methods cannot distinguish between aggregated and free target proteins, easily leading to non-specific degradation of functional free proteins; they cannot achieve early intervention or targeted clearance of stress granules; they cannot be applied to the highly selective degradation of non-enzymatic proteins; the tools have low targeting accuracy, high off-target risk, and insufficient safety; and they lack strategies for precise regulation of the stress granule aggregation process itself, making it impossible to construct pathological stress granule models and functional research tools. Therefore, developing precise regulatory tools with aggregate selective recognition, early intervention capabilities, and compatibility with non-enzymatic core proteins is a pressing technical problem to be solved in this field. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problem of low targeting of existing tools for regulating the degradation of stress particles.

[0007] To address the aforementioned technical problems, this invention provides a fusion protein for targeted degradation of stress particles and its applications. Firstly, this invention provides a fusion protein for targeted degradation of stress particles, which uses G3BP1 protein as a recognition module and TRIM21 as an inducing degradation module, enabling specific degradation of stress particle aggregation. Furthermore, this invention found that using the NTF2L domain of G3BP1 protein as the recognition module and the RING domain of TRIM21 as the inducing degradation module yields the best degradation effect on aggregated stress particles. In summary, this invention provides a fusion protein with aggregate recognition capabilities, inducible ubiquitination modification, and promoted degradation, demonstrating clear practicality, innovation, and scalability in stress particle regulation, research on the pathological mechanisms of stress particle aggregation, and the construction of related disease models.

[0008] The first objective of this invention is to provide a fusion protein for targeted degradation of stress particles, the fusion protein comprising a recognition module and an induced degradation module, wherein the recognition module comprises a G3BP1 protein and the induced degradation module comprises a TRIM21 protein, the amino acid sequence of the G3BP1 protein is shown in SEQ ID NO.1 and the amino acid sequence of the TRIM21 protein is shown in SEQ ID NO.2.

[0009] Furthermore, the fusion protein also includes a fusion tag.

[0010] Furthermore, the fusion tag includes a fluorescent tag, an epitope tag, and / or an affinity purification tag, wherein the fluorescent tag includes, but is not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (mCherry), and blue fluorescent protein (BFP), the epitope tag includes, but is not limited to, the FLAG tag and the HA tag, and the affinity purification tag includes, but is not limited to, the His tag and the GST tag.

[0011] Furthermore, the fusion protein uses the NTF2L domain of the G3BP1 protein as a recognition module and the RING domain of TRIM21 as an induction degradation module, wherein the amino acid sequence of the NTF2L domain is shown in SEQ ID NO.5 and the amino acid sequence of the RING domain is shown in SEQ ID NO.3.

[0012] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.6.

[0013] A second objective of this invention is to provide an application of the above-mentioned fusion protein in the preparation of products that target and degrade stress particles.

[0014] Furthermore, the fusion protein in the product is induced to be expressed using a tetracycline-inducible gene expression system.

[0015] A third objective of this invention is to provide a product that targets and degrades stress particles, the product comprising the aforementioned fusion protein.

[0016] A fourth objective of this invention is to provide the use of the above-described fusion protein or product in a treatment product for neurodegenerative diseases.

[0017] Furthermore, the neurodegenerative diseases include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia.

[0018] A fifth objective of this invention is to provide a method for targeted degradation of stress particles, wherein the method involves expressing the aforementioned fusion protein in target cells containing aggregated stress particles.

[0019] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0020] (1) The fusion protein of the present invention has the ability to recognize and target aggregates: The present invention uses a protein domain with aggregation tendency as an aggregate recognition module, which is located only in the core region of aggregation during the formation of stress particles. It has no obvious binding ability to target proteins in the non-aggregated state and has a high state dependence and specificity.

[0021] (2) Achieving a modular and engineerable degradation strategy: By integrating the identification module with the induced degradation module, this invention achieves accurate identification of stress particle aggregates and ubiquitination-driven degradation, establishes an aggregation-dependent degradation pathway, has a simple construction method, strong adaptability, and is applicable to a variety of expression systems.

[0022] (3) Timing control can be achieved through exogenous induction system: The fusion protein constructed in this invention can be further combined with tetracycline-inducible gene expression system to form a controllable expression system, thereby achieving precise regulation of stress particle formation and intervention time, which is suitable for inducible experimental design and reversible verification of aggregate clearance process.

[0023] (4) Convenient experimental visualization and evaluation: The fusion protein of the present invention can be constructed into a plasmid with a fluorescent tag. Its localization changes and intervention effects can be observed in living cells in real time with the help of fluorescent signals. It can also be quantitatively evaluated by combining various experimental methods such as fluorescence bleaching recovery, protein extraction, and separation of soluble and insoluble proteins. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structural composition of the fusion protein of the present invention;

[0025] Figure 2 This is a schematic diagram of the experimental procedure and results of the fusion protein of the present invention inhibiting the formation of stress particles;

[0026] Figure 3 This is a schematic diagram illustrating the experimental procedure and mechanism for clearing formed protein aggregates according to the present invention;

[0027] Figure 4 The expression plasmid for the fusion protein was constructed using G3BP1 and its functional domains as recognition modules.

[0028] Figure 5 This is a diagram illustrating the modular fusion protein conformation screening and stress particle inhibition effect of the present invention, wherein... Figure 5 In the figure, 'a' represents the immunofluorescence imaging results of different fusion protein expressions, GFP represents green fluorescent protein, and TlA1 represents cytotoxic particle-associated RNA-binding protein 1, and so on. Figure 5 In the table, b represents the statistical result of the proportion of stress granule-positive cells in each group of transfected cells. "****" indicates P<0.0001, and the same applies below.

[0029] Figure 6 The figure shows the experimental results of the expression of the fusion protein NTF2L-RING and its inhibition of stress granule formation. Figure a shows the effect of fusion protein expression on the formation of cellular stress granules, where mCherry represents red fluorescent protein; Figures b and c show the statistical results of the number of stress granules labeled with G3BP1 and UBAP2L (ubiquitin-associated protein 2) in single cells of transfected cells in each group.

[0030] Figure 7 This is a graph showing the selective regulatory effect of the fusion protein NTF2L-RING of the present invention on the condensed state of G3BP1 of stressed particles in a dilution / concentration phase separation system. GAPDH is glyceraldehyde-3-phosphate dehydrogenase, which is an internal control protein.

[0031] Figure 8 This is a graph analyzing the effect of the DCP1A-RING fusion protein on the formation of G3BP1-labeled stress granules;

[0032] Figure 9 The figure shows the experimental results of NTF2L-RING fusion protein expression and its effect on processing bodies. In the figure, a is the laser confocal microscopy image of each group, and b is the percentage of cells in each group that contain more than 6 DCP1A-labeled processing bodies. "ns" indicates P≥0.05, which means that there is no statistically significant difference between groups.

[0033] Figure 10 This is a graph showing the separation results of soluble / insoluble proteins in response to stress particle degradation after NTF2L-RING fusion protein expression. "+" indicates addition, and "-" indicates no addition. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] The proteins and their amino acid sequence numbers involved in the following examples are shown in Table 1.

[0036] Table 1 Proteins and their sequence numbers

[0037]

[0038] The experimental procedures involved in the following embodiments are as follows:

[0039] I. Operational Procedures for Inhibiting Aggregate Formation

[0040] like Figure 2 As shown, target cells were transfected with an empty expression vector plasmid (without recognition and degradation induction modules), a fusion gene expression plasmid (containing recognition and degradation induction modules), and a mutant plasmid lacking degradation induction function (containing a recognition module but lacking the degradation induction module). After 24 hours of culture, cells were subjected to oxidative stress treatment, such as 0.5 mM sodium arsenite for 30 minutes to induce stress granule formation. Cells were then fixed and the core component of the stress granules, G3BP1, was labeled using immunofluorescence staining. Fluorescence microscopy revealed that in cells expressing the fusion protein, the typical stress granules labeled with green G3BP1 were significantly reduced or even completely absent. In contrast, in the control group cells expressing the mutant plasmid lacking degradation induction function, the aggregation core could not be effectively interfered with, and stress granules formed normally. This clearly demonstrates that the fusion protein possesses a clear state selectivity and intervention ability for aggregates, and can inhibit the assembly and stabilization of stress granules through degradation pathways.

[0041] II. Procedures for Clearing Existing Aggregates

[0042] like Figure 3The process involves transfecting target cells with a tetracycline-inducible gene expression plasmid containing the fusion protein sequence, allowing sufficient time to establish the expression system. Subsequently, aggregate formation is induced. This can be achieved by transfecting pathogenic protein expression plasmids with aggregation tendency or overexpressing key assembly proteins derived from the recognition module (such as the stress granule core protein G3BP1) to form pathologically relevant aggregates or stress granule-like structures. For some experimental models, short-term chemical stress (e.g., treatment with 0.5 mM sodium arsenite for 30 minutes) can also be used to assist aggregate formation, suitable for verifying the early expression of the fusion protein. After confirming aggregate formation, doxycycline (Dox) is added to initiate the expression of the fusion protein, enriching it in the subcellular region where the aggregates are located and inducing ubiquitination locally, thereby guiding the aggregates to be removed through the proteasome or other intracellular degradation pathways. Subsequent methods, such as real-time observation under fluorescence microscopy, immunofluorescence staining, fluorescence bleaching recovery assay, separation of soluble and insoluble proteins, and Western blotting, can be used to quantitatively assess the aggregate clearance effect and the intervention capability of the fusion protein.

[0043] The aggregate induction method is not limited to stress conditions and can also include abnormal protein aggregation caused by overexpression of pathology-related proteins, aggregation of RNA-binding proteins, transcription / translation disorders, or specific drug treatments. The recognition module, induction degradation module, and expression regulation module in the fusion protein can be modularly adjusted and replaced according to the type and mechanism of the target aggregate, adapting to different cell models and pathological states. The aggregate intervention strategy provided by this invention has high versatility and is suitable for studying the formation mechanism, dynamic regulation, and clearance process of intracellular protein aggregation. It can also serve as a key technical tool in biomedical research scenarios such as establishing aggregation-related disease models, elucidating mechanisms of action, and drug screening, possessing good experimental adaptability and promising prospects for widespread application.

[0044] Example 1: Construction of a fusion protein expression plasmid for specific degradation of the stress particle core protein G3BP1

[0045] This embodiment designed and constructed a fusion protein expression plasmid containing a recognition module and an induced degradation module (e.g., ...). Figure 1(As shown). This plasmid can express the corresponding fusion protein in eukaryotic cells, achieving specific recognition of G3BP1 and ubiquitin-mediated degradation. In this embodiment, the full-length core protein G3BP1, the NTF2L domain of G3BP1, or the IDR3 domain of G3BP1 are used as modules for recognizing stress granules. The RING domain of the E3 ligase TRIM21, the 26S proteasome subunits PSMC2, PSMC5, or the C-terminal truncated form (CTD) of PSMC5 are used as modules for inducing degradation. Molecular cloning technology is used to fuse the recognition module and the inducing degradation module and construct them into a mammalian expression vector to obtain a fusion protein expression plasmid that can be used for subsequent cell function experiments, thereby achieving the regulation of G3BP1-mediated stress granules (e.g., Figure 2 (As shown).

[0046] (1) G3BP1-RING fusion protein

[0047] Using the full-length human G3BP1 protein as the recognition module and the RING domain of the E3 ubiquitin ligase TRIM21 as the inducible degradation module, the G3BP1-RING fusion sequence was constructed and carried into the eukaryotic expression vector pEGFP-C3 to obtain the pEGFP-C3-G3BP1-RING fusion protein expression plasmid. The preparation method is as follows:

[0048] Total RNA was extracted from HEK293 cells, and cDNA libraries were obtained by reverse transcription. Targeting the RING domain of G3BP1 protein and TRIM21, PCR primers were designed with a HindIII restriction enzyme site at the 5' end of G3BP1 and an EcoRI restriction enzyme site at the 3' end, derived from Escherichia coli. Similarly, the RING fragment had an EcoRI restriction site at the 5' end and a BamHI restriction enzyme site at the 3' end. G3BP1 and RING fragments were amplified using high-fidelity DNA polymerase and purified after 1% agarose gel electrophoresis. The purified G3BP1 and RING fragments were double-digested and ligated using T4 DNA ligase to obtain the G3BP1-RING fragment, which has a HindIII restriction enzyme site at the 5' end and a BamHI restriction enzyme site at the 3' end. The pEGFP-C3 vector was double-digested with HindIII and BamHI to linearize the vector. The G3BP1-RING fragment and the pEGFP-C3 vector were ligated using T4 DNA ligase and transformed into DH5α competent E. coli. Positive clones were selected, and the plasmid was extracted and the sequence correctness and ligation direction were verified by dideoxy sequencing (Sanger sequencing).

[0049] Table 2 Primers used for the construction of the G3BP1-RING fusion protein

[0050]

[0051] (2) NTF2L-RING fusion protein

[0052] The NTF2L-RING fusion protein uses the NTF2L domain of the truncated G3BP1 protein as the recognition module and the RING domain of the E3 ubiquitin ligase TRIM21 as the inducible degradation module. The NTF2L-RING fusion sequence was constructed and carried into the eukaryotic expression vector pEGFP-C3, resulting in the expression plasmid pEGFP-C3-NTF2L-RING. The preparation method is as follows:

[0053] Using the NTF2L domain of G3BP1 as the target, primers were designed using pEGFP-C3-G3BP1-RING constructed in (1) as a template. The NTF2L fragment and vector were obtained by PCR and purified by 1% agarose gel electrophoresis. The purified NTF2L fragment and vector were recombined with homologous recombinase and transformed into DH5α competent Escherichia coli. Positive clones were picked, plasmids were extracted, and the sequence correctness and ligation direction were verified by Sanger sequencing.

[0054] Table 3 Primers used for the construction of the NTF2L-RING fusion protein

[0055]

[0056] (3) IDR3-RING fusion protein

[0057] Using the IDR3 domain of the truncated G3BP1 protein as the recognition module and the RING domain of the E3 ubiquitin ligase TRIM21 as the inducible degradation module, the IDR3-RING fusion sequence was constructed and carried into the eukaryotic expression vector pEGFP-C3 to obtain the pEGFP-C3-IDR3-RING fusion protein expression plasmid. The preparation method is as follows:

[0058] Using the IDR3 domain of G3BP1 as the target, primers were designed using pEGFP-C3-G3BP1-RING constructed in (1) as a template. The IDR3 fragment and vector were obtained by PCR and purified by 1% agarose gel electrophoresis. The purified IDR3 fragment and vector were recombined with homologous recombinase and transformed into DH5α competent Escherichia coli. Positive clones were picked, plasmids were extracted, and the sequence correctness and ligation direction were verified by Sanger sequencing.

[0059] Table 4 Primers used for the construction of the IDR3-RING fusion protein

[0060]

[0061] (4) G3BP1-PSMC2 fusion protein

[0062] Using the full-length human G3BP1 protein as the recognition module and the proteasome subunit PSMC2 as the inducible degradation module, a fusion sequence of G3BP1-PSMC2 was constructed and carried into the eukaryotic expression vector pEGFP-C3 to obtain the fusion protein expression plasmid pEGFP-C3-G3BP1-PSMC2. The preparation method is as follows:

[0063] Using the cDNA library obtained in (1) as a template, primers were designed with human 26S proteasome subunit PSMC2 as the target, and the PSMC2 fragment was obtained by PCR. Using pEGFP-C3-G3BP1-RING constructed in (1) as a template, primers were designed, and the vector was obtained by PCR. After 1% agarose gel electrophoresis, the vector was recovered and purified. The recovered and purified PSMC2 fragment and the vector were recombined with homologous recombinase and transformed into DH5α competent Escherichia coli. Positive clones were picked, plasmids were extracted, and the sequence correctness and ligation direction were verified by Sanger sequencing.

[0064] Table 5 Primers used for the construction of the G3BP1-PSMC2 fusion protein

[0065]

[0066] (5) G3BP1-PSMC5 fusion protein

[0067] Using the full-length human G3BP1 protein as the recognition module and the human 26S proteasome subunit PSMC5 as the inducible degradation module, a fusion sequence of G3BP1-PSMC5 was constructed and carried into the eukaryotic expression vector pEGFP-C3 to obtain the fusion protein expression plasmid pEGFP-C3-G3BP1-PSMC5. The preparation method is as follows:

[0068] Total RNA was extracted from human HEK293 cells, and a cDNA library was obtained by reverse transcription. Primers were designed targeting the human 26S proteasome subunit PSMC5, and the PSMC5 fragment was obtained by PCR. Using pEGFP-C3-G3BP1-RING constructed in (1) as a template, primers were designed, and the vector was obtained by PCR. After 1% agarose gel electrophoresis, the fragment was recovered and purified. The recovered and purified PSMC5 fragment and the vector were recombined with homologous recombinase and transformed into DH5α competent Escherichia coli. Positive clones were picked, plasmids were extracted, and the sequence correctness and ligation direction were verified by Sanger sequencing.

[0069] Table 6 Primers used for the construction of the G3BP1-PSMC5 fusion protein

[0070]

[0071] (6) G3BP1-PSMC5 CTD fusion protein

[0072] Using the full-length human G3BP1 protein as the recognition module and the C-terminal truncated functional domain (CTD) of the human 26S proteasome subunit PSMC5 as the inducible degradation module, a fusion sequence of G3BP1-PSMC5 CTD was constructed and carried into the eukaryotic expression vector pEGFP-C3 to obtain the fusion protein expression plasmid pEGFP-C3-G3BP1-PSMC5 CTD. The preparation method is as follows:

[0073] Using the CTD of PSMC5 as the target, and pEGFP-C3-G3BP1-PSMC5 constructed in (5) as the template, primers were designed, and the PSMC5 CTD fragment and vector were obtained by PCR. After 1% agarose gel electrophoresis, the fragments were recovered and purified. The recovered and purified PSMC5 CTD fragment and vector were recombined with homologous recombinase and transformed into DH5α competent Escherichia coli. Positive clones were picked, plasmids were extracted, and the sequence correctness and ligation direction were verified by Sanger sequencing.

[0074] Table 7 Primers used for constructing the G3BP1-PSMC5 CTD fusion protein

[0075]

[0076] (7) NTF2L-RING I18R fusion protein (with NTF2L-RING M72E fusion protein as control)

[0077] Specific primers containing mutant sequences were designed for key functional sites of the RING domain in TRIM21. The isoleucine at position 18 of the RING domain was mutated to arginine (RING I18R), and the methionine at position 72 was mutated to glutamic acid (RING M72E). Using the validated pEGFP-C3-NTF2L-RING recombinant plasmid as a template, primers were designed, and PCR was performed to obtain the fragment and vector. After purification by 1% agarose gel electrophoresis, the purified fragment and vector were recombined with homologous recombinase and transformed into DH5α competent E. coli. Positive clones were selected, and plasmids were extracted to obtain the RING domain loss-of-function mutant plasmids pEGFP-C3-NTF2L-RING I18R and pEGFP-C3-NTF2L-RING M72E, which were confirmed by Sanger sequencing and used for subsequent control experiments.

[0078] Table 8 Primers used for the construction of NTF2L-RING I18R and NTF2L-RING M72E fusion proteins.

[0079]

[0080] Based on this, the pEGFP-C3 vector was replaced with the pmCherry-C1 vector to obtain pmCherry-C1-NTF2L-RING, pmCherry-C1-NTF2L-RING I18R and pmCherry-C1-NTF2L-RING M72E.

[0081] Based on this, the NTF2L domain of G3BP1 can be replaced with the IDR3 domain. Following the plasmid construction method described above, pEGFP-C3-IDR3-RING recombinant plasmid that has been verified to be correct can be used as a template to obtain pEGFP-C3-IDR3-RING I18R, pEGFP-C3-IDR3-RING M72E, pmCherry-C1-IDR3-RING, pmCherry-C1-IDR3-RING I18R, and pmCherry-C1-IDR3-RING M72E.

[0082] Example 2: Comparison of expression of fusion proteins with different module combinations and their ability to inhibit stress granule formation

[0083] This embodiment aims to verify the feasibility of the proposed "identification module + induced degradation module" fusion protein design strategy and its intervention ability in stress particle formation through systematic screening of multiple module combinations.

[0084] The epithelioid cell line (HEK293 cells) derived from embryonic kidney cells was seeded in 24-well plates. When the cell density reached approximately 70%, the cells were transfected with plasmids according to the following groups:

[0085] Empty vector control group: Contains plasmid pEGFP-C3;

[0086] Group G3BP1-RING: Contains plasmid pEGFP-C3-G3BP1-RING;

[0087] NTF2L-RING group: contains plasmid pEGFP-C3-NTF2L-RING;

[0088] IDR3-RING group: contains plasmid pEGFP-C3-IDR3-RING;

[0089] Group G3BP1-PSMC2: Contains plasmid pEGFP-C3-G3BP1-PSMC2;

[0090] Group G3BP1-PSMC5: Contains plasmid pEGFP-C3-G3BP1-PSMC5;

[0091] Group G3BP1-PSMC5 CTD: Contains plasmid pEGFP-C3-G3BP1-PSMC5 CTD.

[0092] Twenty-four hours after transfection, each group of cells was treated with 0.5 mM sodium arsenite for 30 minutes and then fixed. The cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% polyethylene glycol tert-octylphenyl ether (Triton X-100) for 8 minutes. Immunofluorescence staining was performed with anti-TIA1 anti-rabbit antibody (dilution ratio 1:1000). The secondary antibody was anti-rabbit immunoglobulin G (IgG) labeled with dibenzocyclooctylene (Alexa Fluor 647) (dilution ratio 1:600). The nuclei were stained with Hoechst nuclear labeling dye (Hoechst 33342, dilution ratio 1:2000) for 20 minutes.

[0093] Figure 5 The results of the assays on the inhibitory effects of different groups on stress granule formation in HEK293 cells were presented. Figure 5 The data presented are statistical values ​​of 81-158 cell samples from 4-9 fields of view in each group, expressed as mean ± standard deviation (Mean ± SD); one-way ANOVA was used for statistical analysis. The results showed that the proportion of cells containing stress granules in the NTF2L-RING group was significantly lower than that in other groups.

[0094] This embodiment demonstrates that various module combinations can reduce stress particle formation to varying degrees, indicating that the modular strategy of "recognition module + induced degradation module" has a certain degree of versatility. The empty control group showed typical large-scale generation of stress particles, while the NTF2L-RING group had the lowest proportion of positive cells expressing stress particles (SGs), indicating that the fusion protein in this group had the best inhibitory effect on sodium arsenite-induced cellular stress particle generation. The fusion proteins in the G3BP1-PSMC2 group and the G3BP1-PSMC5CTD group also showed a certain degree of inhibitory activity on stress particle generation, but the inhibition efficiency was lower than that of the NTF2L-RING group.

[0095] In summary, this embodiment, through systematic screening of various combinations of recognition modules and induced degradation modules, confirmed that the fusion protein (NTF2L-RING) constructed using the NTF2L domain of G3BP1 as the recognition module and the RING domain of TRIM21 as the induced degradation module has the best stress particle inhibition activity.

[0096] Example 3: Expression of fusion protein NTF2L-RING and inhibition of stress granule formation.

[0097] This embodiment aims to verify the functional effectiveness of the "recognition module + induced degradation module" fusion protein design strategy in the regulation of stress particles, and to further confirm whether the inhibitory effect of the preferred fusion protein scheme NTF2L-RING obtained in Example 2 on stress particle formation depends on the functional integrity of the RING domain of the induced degradation module. To this end, the relationship between the structural integrity and functional activity of the fusion protein was systematically evaluated by constructing and comparing the effects of the NTF2L-RING fusion protein, the recognition module-only expression control, and the RING domain functional defect mutant on stress particle formation under oxidative stress conditions.

[0098] HEK293 cells were seeded in 24-well plates. When the cell density reached approximately 70%, they were divided into five groups and transfected with plasmids:

[0099] Empty control group: containing plasmid pmCherry-C1;

[0100] NTF2L-RING group: contains plasmid pmCherry-C1-NTF2L-RING;

[0101] NTF2L group: contains plasmid pmCherry-C1-NTF2L;

[0102] NTF2L-RING M72E group: contains plasmid pmCherry-C1-NTF2L-RING M72E;

[0103] The NTF2L-RING I18R group contains the plasmid pmCherry-C1-NTF2L-RING I18R.

[0104] 24 hours after transfection, each group of cells was treated with 0.5 mM sodium arsenite for 30 minutes and then fixed. The cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 8 minutes. Immunofluorescence staining was performed with anti-G3BP1 anti-mouse antibody (dilution ratio 1:1000). The secondary antibody was Alexa Fluor 488-labeled anti-mouse IgG (dilution ratio 1:600). Immunofluorescence staining was performed with anti-ubiquitin-associated protein 2 (UBAP2L) anti-rabbit antibody (dilution ratio 1:600). The secondary antibody was Alexa Fluor 647-labeled anti-rabbit IgG (dilution ratio 1:600). The nuclei were stained with Hoechst 33342 (dilution ratio 1:2000) nuclear labeling dye for 20 minutes.

[0105] The results are as follows Figure 6As shown, the number of stress granules labeled with G3BP1 and UBAP2L in the NTF2L-RING group was significantly reduced compared with the other four groups. The number of stress granules in the NTF2L group, NTF2L-RING M72E group, and NTF2L-RING I18R group was not significantly different from that in the empty vector control group. This indicates that the NTF2L domain alone or the RING mutant with functional defects cannot inhibit the formation of stress granules. The significant inhibitory effect of the NTF2L-RING group depends on the complete structure and function of the fusion protein, indicating that the expression of fusion protein granules can effectively inhibit the formation of oxidative stress-induced protein aggregates.

[0106] Example 4: Selective regulation of G3BP1 in stress particle condensates by NTF2L-RING fusion protein during dilution / concentration phase separation

[0107] To further verify whether the NTF2L-RING fusion protein has the ability to selectively intervene in target aggregates in a condensed state, this embodiment uses a dilution phase / concentration phase separation system to analyze the phase distribution changes of the stress particle core protein G3BP1 under different experimental conditions.

[0108] HEK293 cells were divided into four groups and transfected with plasmids respectively:

[0109] Empty vector control group: Contains plasmid pEGFP-C3;

[0110] NTF2L-RING group: contains plasmid pEGFP-C3-NTF2L-RING;

[0111] NTF2L group: contains plasmid pEGFP-C3-NTF2L;

[0112] NTF2L-RING I18R: Contains plasmid pEGFP-C3-NTF2L-RING I18R.

[0113] Twenty-four hours after transfection, the cells were treated with 0.5 mM sodium arsenite for 30 minutes to induce stress particle formation.

[0114] After processing, following the dilution / concentration phase separation procedure, cells were collected into centrifuge tubes using phosphate-buffered saline (PBS). The culture medium was removed by centrifugation at 1000 rpm for 5 minutes, and the supernatant was discarded. 200 μL of cell lysis buffer was added, and the cells were thoroughly mixed and sonicated. The cells were then centrifuged at 4°C and 21000 rcf for 15 minutes. The first 100 μL of supernatant was collected as the dilution phase (S) fraction. The remaining precipitate was added to the lysis buffer, mixed, and centrifuged at 21000 rcf for 20 minutes. The washing was repeated twice, and the supernatant was discarded. The cells were then added to the loading buffer, thoroughly mixed, and boiled for denaturation to obtain the concentrated phase (P) fraction. Immunoblot analysis was performed using anti-G3BP1 antibody and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. The specific steps are as follows: after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the gel is transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skim milk powder at room temperature for 1 hour; anti-G3BP1 anti-mouse antibody (1:800) and anti-GAPDH antibody (dilution ratio 1:8000) are added and incubated overnight at 4°C; after washing with tris(hydroxymethyl)aminomethane buffer (TBST), horseradish peroxidase (HRP) labeled secondary antibody (dilution ratio 1:10000) is added and incubated at room temperature for 1 hour; the distribution of G3BP1 in each phase component is detected by chemiluminescence imaging.

[0115] Experimental results are as follows Figure 7 As shown, in the control group, NTF2L group, and NTF2L-RING I18R group, G3BP1 was mainly distributed in the dilute phase (S) fraction, and a certain amount of G3BP1 signal could also be detected in the condensed phase (P). However, in cells expressing the NTF2L-RING fusion protein, the intensity of the G3BP1 band in the condensed phase (P) fraction was significantly lower than that in other groups. This indicates that the NTF2L-RING fusion protein can significantly reduce the content of condensed (condensed) G3BP1, while the expression of the recognition module alone or the induction of the degradation module inactivation could not produce the same effect. These results further demonstrate that the NTF2L-RING fusion protein has the ability to selectively regulate the state of condensed target proteins in cell phase separation systems.

[0116] Example 5: Validation of the targeting specificity of the NTF2L-RING fusion protein

[0117] (1) Analysis of the role of fusion proteins constructed from different recognition modules in the formation of stress granules

[0118] To verify whether the NTF2L-RING fusion protein exhibits target specificity in its intracellular function, this embodiment selected uncapped mRNA1A (DCP1A), a protein located in the cytoplasmic processing body (P-body), a membraneless organelle structure independent of stress granules, as the recognition module, and RING of TRIM21 as the inducible degradation module. The DCP1A-RING fusion protein was constructed, and its effect on the aggregation state of the stress granule marker protein G3BP1 was examined, thereby verifying the target specificity of the fusion protein. The specific methods are as follows:

[0119] Using the cDNA library obtained in Example 1 (1) as a template, primers were designed with human DCP1A as the target, and the DCP1A fragment was obtained by PCR. Using the correctly sequenced pEGFP-C3-G3BP1-RING constructed in Example 1 as a template, primers were designed, and the vector was obtained by PCR. After 1% agarose gel electrophoresis, the vector was recovered and purified. The recovered and purified DCP1A fragment and the vector were recombined with homologous recombinase and transformed into DH5α competent Escherichia coli. Positive clones were picked, plasmids were extracted, and the sequence correctness and ligation direction were verified by Sanger sequencing to obtain the pEGFP-C3-DCP1A-RING fusion protein expression plasmid.

[0120] Table 9 Primers used for the construction of the DCP1A-RING fusion protein

[0121]

[0122] HEK293 cells were seeded in 24-well plates. When the cell density reached approximately 70%, cells were transfected with pEGFP-C3 plasmid as the empty vector control group and pEGFP-C3-DCP1A-RING plasmid as the DCP1A-RING group. Twenty-four hours after transfection, cells were treated with 0.5 mM sodium arsenite for 30 minutes to induce stress granule formation. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 8 minutes, and immunofluorescence staining was performed using anti-G3BP1 anti-rabbit antibody (dilution 1:1000). Detection was performed using Alexa Fluor 594-labeled anti-rabbit secondary antibody (dilution 1:600). Microscopic imaging was used to statistically analyze the number and intensity of G3BP1 positive aggregates and the proportion of positive stress granules in the cells.

[0123] Experimental results are as follows Figure 8 As shown, compared with the empty control group, there were no significant changes in the number of G3BP1-labeled stress particles, aggregation intensity, and proportion of positive cells in the DCP1A-RING group. This result indicates that the fusion protein constructed using P-body protein DCP1A as the recognition module did not have a detectable effect on stress particle formation.

[0124] (2) Analysis of the effect of NTF2L-RING fusion protein on processing bodies

[0125] In this embodiment, the NTF2L-RING fusion protein was expressed, and the effect of the fusion protein on DDX6 (dead-box helicase 6), a key component of the processing body, was observed using immunofluorescence detection. This study verified the targeting effect of the NTF2L-RING fusion protein as a tool protein on stress particles.

[0126] Epithelial-like cells (HEK293 cells) derived from human embryonic kidney cells were seeded in 24-well plates. When the cell density reached approximately 70%, the cells were transfected with the following 5 groups of plasmids:

[0127] Empty vector control group: Contains plasmid pEGFP-C3;

[0128] NTF2L-RING group: contains plasmid pEGFP-C3-NTF2L-RING;

[0129] NTF2L group: contains plasmid pEGFP-C3-NTF2L;

[0130] NTF2L-RING M72E group: contains plasmid pEGFP-C3-NTF2L-RING M72E;

[0131] NTF2L-RING I18R group: contains plasmid pEGFP-C3-NTF2L-RING I18R.

[0132] 24 hours after transfection, each group of cells was treated with 0.5 mM sodium arsenite for 30 minutes and then fixed. The cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 8 minutes. Immunofluorescence staining was performed with anti-dead box helicase 6 (DDX6) anti-mouse antibody (dilution ratio 1:600). The secondary antibody was Alexa Fluor 594-labeled anti-mouse IgG (dilution ratio 1:600). The nuclei were stained with Hoechst 33342 (dilution ratio 1:2000) nuclear labeling dye for 20 minutes.

[0133] The results are as follows Figure 9As shown, the percentage of cells expressing the NTF2L-RING fusion protein containing more than 6 processing bodies did not show a statistically significant difference compared to the empty vector control group, suggesting that the expression of the NTF2L-RING fusion protein did not have a detectable effect on the formation and distribution of processing bodies. This result further indicates that the NTF2L-RING fusion protein does not exert broad, non-selective intervention on non-membrane organelles, but rather exhibits targeting characteristics against stress granules under specific conditions. In summary, this embodiment confirms that the function of the NTF2L-RING fusion protein depends on the target specificity of the recognition module. The intervention of the NTF2L-RING fusion protein on SGs is not due to non-specific protein degradation induced by the RING domain, but is driven by the selective binding of the recognition module to specific condensates.

[0134] Example 6: Construction of NTF2L-RING fusion protein expression plasmid regulated by the tetracycline regulatory system (Tet-On system)

[0135] This embodiment is based on the existing tetracycline-inducible expression plasmid TetOn-mCherry. By cloning the NTF2L-RING fusion fragment to the C-terminus of mCherry, a TetOn-mCherry-NTF2L-RING fusion protein plasmid that can be inducibly expressed in mammalian cells is constructed.

[0136] Using the NTF2L-RING fragment constructed and verified correctly in Example 1 as a template, the target fragment and vector were amplified by PCR. After homologous recombination, the purified vector and fragment were recovered by 1% agarose gel electrophoresis. The homologous recombination product was transformed into DH5α competent cells, and positive clones were selected. Plasmid extraction and Sanger sequencing verified that the NTF2L-RING fragment was correctly fused downstream of mCherry and that the sequence was correct, resulting in the TetOn-mCherry-NTF2L-RING recombinant plasmid that can be induced to express. This construction, while retaining the fluorescence function of mCherry, allows the expression of the fusion protein to be induced by exogenous doxycycline (DOX, a derivative of tetracycline, which is often used in experiments to replace tetracycline for induction), achieving precise control over the spatiotemporal expression of the fusion protein for subsequent studies on the intervention function of protein aggregates.

[0137] Table 10 Primers used for constructing the mCherry-NTF2L-RING fusion protein expression plasmid

[0138]

[0139] HEK293 cells were seeded in 12-well plates until the cell density reached approximately 70%. Simultaneously, the cells were transfected with the Tet-On system-regulated mCherry-labeled fusion protein expression plasmid (TetOn-mCherry-NTF2L-RING) and the aggregation-inducing plasmid GFP-G3BP1. 12-24 hours after transfection, once aggregates formed within the cells, doxycycline (1 μg / mL) was added to the culture medium to induce fusion protein expression. 24 hours after induction, cell samples were collected, and soluble and insoluble proteins were separated. The cell suspension was transferred to centrifuge tubes with phosphate-buffered saline and centrifuged at 5000 rpm for 3 minutes, discarding the supernatant. Add 250 μL of soluble fraction lysis buffer to each sample (formulation: 500 μL of 1M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.4), 500 μL of Triton X-100, 500 μL of 1.5M NaCl, 5 mL of glycerol, and the remainder deionized water). Mix thoroughly by pipetting to disperse cell clumps and place on ice. Tap the centrifuge tube lightly every 20 minutes for approximately 1 hour. Centrifuge the samples at 13500 rpm for 20 minutes at 4°C. Transfer 100 μL of the supernatant to a new centrifuge tube as the soluble fraction. The precipitate is the insoluble fraction; add 100 μL of soluble fraction lysis buffer, centrifuge at 13500 rpm for 20 minutes, and repeat the washing process twice. Discard the supernatant, add 100 μL of insoluble fraction lysis buffer (formulation: take 10 mL of the above soluble fraction lysis buffer, add 0.4 g of sodium dodecyl sulfate (SDS) to a final concentration of 4%, dissolve and mix well), sonicate, add 100 μL of loading buffer, and mix gently. Place the sample in boiling water for 8 minutes. After boiling, store the sample in a -20°C freezer. Subsequently, use anti-G3BP1 antibody and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for Western blotting to analyze the distribution changes of G3BP1 in soluble and insoluble fractions.

[0140] Test results as follows Figure 10 As shown, in the stress particle model formed by GFP-G3BP1 overexpression, the induced expression of the NTF2L-RING fusion protein significantly reduced the proportion of insoluble G3BP1 without affecting soluble G3BP1. These results indicate that the NTF2L-RING fusion protein can selectively recognize and promote the degradation of stress particles without affecting soluble proteins, thereby achieving specific targeted clearance of stress particles.

[0141] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A fusion protein that targets and degrades stress particles, characterized in that, The fusion protein includes a recognition module and an induced degradation module, wherein the recognition module includes GTPase activator protein SH3 domain binding protein 1, and the induced degradation module includes E3 ubiquitin ligase. The amino acid sequence of the GTPase activator protein SH3 domain binding protein 1 is shown in SEQ ID NO.1, and the amino acid sequence of the E3 ubiquitin ligase is shown in SEQ ID NO.

2.

2. The fusion protein according to claim 1, characterized in that, The fusion protein also includes a fusion tag.

3. The fusion protein according to claim 1, characterized in that, The fusion protein uses the SH3 domain of the GTPase activator protein to bind the NTF2L domain of protein 1 as a recognition module and the RING domain of the E3 ubiquitin ligase as an induction degradation module. The amino acid sequence of the NTF2L domain is shown in SEQ ID NO.5, and the amino acid sequence of the RING domain is shown in SEQ ID NO.

3.

4. The fusion protein according to claim 3, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

6.

5. The use of the fusion protein according to any one of claims 1-4 in the preparation of products that target and degrade stress particles.

6. The application according to claim 5, characterized in that, The fusion protein in the product is induced to be expressed using a tetracycline-inducible gene expression system.

7. A product for targeted degradation of stress particles, characterized in that, The product includes the fusion protein according to any one of claims 1-4.

8. The use of the fusion protein according to any one of claims 1-4 or the product according to claim 7 in a product for the treatment of neurodegenerative diseases.

9. The application according to claim 8, characterized in that, The neurodegenerative diseases mentioned include amyotrophic lateral sclerosis (ALS) or frontotemporal dementia.

10. A method for targeted degradation of stress particles, characterized in that, The method involves expressing the fusion protein according to any one of claims 1-4 in target cells, wherein the target cells contain aggregated stress granules.

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